PROJECT SUMMARY Pulmonary hypertension (PH) is a progressive, life-threatening disease that often develops secondary to the chronic hypoxia of cardiopulmonary disease or high-altitude (HA) residence (? 2500m). Over the past few decades, PH-associated mortality has increased in both sexes of all race and ethnic groups, and no current therapy has proven effective for hypoxia-induced PH. Adverse intrauterine conditions, including fetal growth restriction (IUGR), preeclampsia and perinatal hypoxia, can induce durable changes to the structure and function of the lung and pulmonary vasculature, and are predictive of pulmonary vascular disease in affected offspring. Our overarching goal is to determine whether intrauterine growth restriction (IUGR) increases susceptibility to hypoxia-induced pulmonary hypertension by altering the epigenetic regulation of genes belonging to the mTOR pathway. To address this goal, we propose to conduct three integrated scientific aims. In Aim 1, we will define the effect of IUGR on mTOR pathway DNAm and gene expression patterns, and mTOR signaling in PBMCs and two representative vascular cell types (HUAECs and HUASMCs) at birth in humans using both primary and external validation cohorts. Our primary cohort will be infants born at LA or HA in Bolivia (400 and 3600 m), and our external validation cohort will consist of infants born in Frisco, Colorado to women living ? 3000m. Obtaining samples from Bolivia for our primary data set is beneficial because the La Paz metropolitan region comprises the largest, HA resident population in the world with more than two million persons living ? 3000m and the modern medical and research facilities necessary to conduct the proposed study aims; this will improve our efficiency with respect to obtaining a sufficient number of subjects for prospective study over the proposed timeframe. In Aim 2, we will establish the relationship between mTOR DNAm and expression patterns at birth and echocardiographic indices of PH measured prospectively across the first year of life (1 week, 6 and 12 months). Because DNAm marks are influenced by environmental and genetic factors, we will also determine whether differentially methylated mTOR pathway genes in cases of IUGR and/or PH during infancy, are associated with a) SNPs neighboring (< 500 kb) differential methylation marks, or b) SNPs within genes encoding proteins known to be involved in PH. Using this approach, we will determine the interactive effect of genetic factors, epigenetic marks and IUGR for PH. In Aim 3 we will use genetic and pharmacologic approaches in an established murine model of hypoxia-induced IUGR and PH to study the interaction of DNAm state, mTOR pathway signaling and IUGR for the developmental programming of pulmonary vascular dysfunction. Together, our research aims 1) address major knowledge gaps with respect to the mechanisms underlying the effect of intrauterine exposures to compromise the pulmonary circulation and 2) have the potential to identify targets for new therapeutic or preventive strategies to reduce the burden of pulmonary vascular disease across the lifespan.